1. Technical Field
This invention relates to the field of TGF-beta factor purification from a dairy source.
2. Description of Prior Art
Milk, in particular human and mammal milk, contains several bioactive polypeptides, especially numerous growth factors. One of these growth factors contained in milk is TGF-beta (Transforming Growth Factor beta).
The term TGF-beta designates a family of different growth factors. TGF-beta 1 and TGF-beta 2 are two homologous forms of TGF-beta. They are homodimeric constituents made up of two similar polypeptide chains each containing 112 amino-acids, linked by a disulfide bridge. Their molecular mass is 25,000 Daltons. TGF-beta 1 and TGF-beta 2 have 72% of structural homology and they present similar biological properties. Bovine and Human TGF-beta 2 are identical in respect of their amino-acid sequence.
TGF-beta can be obtained by genetic recombination in purified form. But preparations containing TGF-beta recombinants are susceptible to contain some bacterial proteins, of which some are allergenic. In addition, by definition, purified preparations of TGF-beta recombinants do not contain associated milk proteins which can complete or increase the TGF-beta biological effect.
Cow's milk contains both TGF-beta 1 or TGF-beta 2. TGF-beta 2 is the main component and represents 90% in weight of TGF-beta found in milk, while TGF-beta 1, on the other hand, represents 10% in weight of the total TGF-beta content in milk.
In milk, more than 90% of the TGF-beta 2 is in latent form, i.e. in a non-biologically active form.
Various scientific studies have shown that TGF-beta content in milk is from 12 to 150 μg/l in colostrum, from 3.7 to 3.8 μg/l in crude and pasteurized milk, 4.3 μg/l in skimmed milk, and 3.7 μg/l in whey.
Biological activities of TGF-beta are numerous, which give to this polypeptide a great therapeutical interest for prevention or treatment of a large variety of diseases or pathologies.
TGF-beta is biologically active on the extra-cellular matrix. It stimulates the synthesis of matrix proteins and increases the synthesis of collagen and fibronectin in fibroblasts. It also has an inhibitory effect on the synthesis of proteolytic enzymes such as collagenase and metalloproteases. TGF-beta increases the secretion of protease inhibitors such as the plasminogen activator inhibitor or metalloprotease inhibitors.
TGF-beta has also a biologic activity on the skeleton. In particular, TGF-beta is in high concentration in bones. It has an activity on cartilage formation, stimulates the resorption of osteoclasts, and the activation of osteoblasts. It acts as a natural inhibitor of the resorption of bones and provides bone formation stimulation.
TGF-beta is also active towards lymphocytes. By way of illustration, it inhibits T-lymphocytes proliferation and contributes to the activity of so-called “Natural Killers” cells.
TGF-beta is also a powerful anti-inflammatory agent. It decreases pro-inflammatory cytokines production. It has immunosuppresive properties and inhibits the proliferation of activated T-lymphocytes.
In addition, TGF-beta has antiproliferative effects. It is a strong inhibitor of epithelial cells. TGF-beta has a strong anti-mitogenic activity towards mesenchym cells, embryonic fibroblasts, endothelial cells, and T and B lymphocytes. It also has a strong inhibitory effect on the growth of hepatocytes, and could possibly have an important role in maintaining the quiescent state of thereof. TGF-beta also acts as a negative regulation factor of the mammary epithelium.
TGF-beta also has anticancer effects. During carcinogenesis, cancer cells can loose their ability to respond to TGF-beta. Nevertheless, some epithelial cell tumors, like breast cancer cells, are sensitive to anti-proliferative effects of TGF-beta. Such is the case for breast cancer cells.
TGF-beta also acts on proliferation and differentiation of leukemia cells. It inhibits the proliferation of promyelocyte cells. It could have a synergistic effect with retinoic acid and vitamin D3.
European patent application No. EP 0 527.283 in the name of Société des Produits NESTLE S.A. describes a process for preparing a milk-derived product containing TGF-beta. During this process, the crude milk is skimmed by centrifugation, desalted on a PD-10 (Pharmacia) chromatographic column, and then sterilized by filtration on a “Millipore” membrane with a 0.2 μm pore diameter. The skimmed milk is sterilized, adjusted at pH 4.0 with 1N HCl, and then centrifuged at 40,000 g during 60 minutes to separate precipitated casein from whey. The separated whey is neutralized by 1N sodium hydroxide and dialyzed. However, this process, which is able to eliminate casein from the initial skimmed milk, is not a process to purify TGF-beta. In fact, the final product contains all the whey proteins from the initial milk, where the TGF-beta is, but without significant enrichment of this specific protein.
In the state of the art, many processes of obtaining protein fractions enriched in TGF-beta from milk have been described.
European patent application EP 0 313.515 in the name of CIBA GEIGY describes a process for purification of a growth factor contained in milk, with successive chromatographic steps, especially cation exchange resins, hydrophobic interaction chromatography (RP-HPLC) or size exclusion chromatography supports.
The PCT application No. WO 01 25.276 in the name of CAMPINA MELKUNIE B.V. describes a process for extracting TGF-beta and similar to insulin growth factors (IGF-1) from a dairy product. This process comprises the following steps:
a) recovering a base fraction of the dairy product by cation exchange chromatography;
b) passing the fraction obtained in step a) on a hydroxyapatite column; and
c) elution of the hydroxyapatite column with suitable eluents selected so as to obtain for example a TGF-beta fraction substantially free of IGF-1.
All the processes described above have technical disadvantages. As a matter of fact, TGF-beta purification processes involving successive chromatographic steps are long and tedious. The important number of chromatographic steps necessary for achieving a desired degree of purity considerably decreases the final yield because of the progressive TGF-beta degradation while purification is carrying out, and the unavoidable loss of biologically active TGF-beta at each of the chromatography steps. In addition to the use of different saline and highly polluting regeneration solutions that must thereafter be eliminated, these processes present a high risk of bacterial contamination of the final product.
There is therefore a need in the state of the art for improved processes allowing the purification of TGF-beta from a dairy product, without the disadvantages mentioned above of the processes currently available in the art.